In large animal models of hypoxic pulmonary hypertension (PHTN) that closely parallel human disease, the earliest pulmonary artery (PA) smooth muscle (SMC) structural changes occur at the medial/adventitial border. Distal muscularization of vessels is also a prominent feature. Mechanisms that regulate the accumulation of SMCs in remodeled pulmonary vessels are still unclear. Susceptibility to apoptosis is likely an important determinant. Neprilysin (NEP) is a key cell surface peptidase involved in the degradation of vasoactive neuropeptides. Less well appreciated is the emerging concept that NEP may also influence cell responses by directly engaging in intracellular signaling through novel peptidase-independent mechanisms. NEP has recently been implicated in apoptosis of cancer cells. These observations support the possibility that NEP could exert a protective effect against hypoxic PHTN by increasing susceptibility of PA SMC to hypoxia-induced apoptosis. Loss of NEP would then predispose to exaggerated pulmonary vascular remodeling in response to hypoxia by protecting PA SMC from apoptosis. We now have data to support this concept. We have found that deletion of NEP in mice predisposes to exaggerated hypoxic PHTN. Lungs and isolated PA SMC from NEP null mice have decreased susceptibility to hypoxia-induced apoptosis compared to wild type controls. NEP replacement increases susceptibility of NEP null PA SMC to hypoxia-induced apoptosis. Protection from apoptosis in the absence of NEP is associated with up-regulation of apoptosis-associated kinases and stress proteins. We also have evidence that NEP may be interacting nonenzymatically through its cytoplasmic tail with multiple as yet unidentified proteins. The following Hypotheses will be tested: #1) NEP protects the lung vasculature from the development of hypoxic PHTN and limits vascular remodeling at least in part by increasing susceptibility of PA SMC to hypoxia-induced apoptosis; #2) NEP increases susceptibility of PA SMC to hypoxia-induced apoptosis by decreasing activity of selected kinases (focus: PI3 kinase, Akt, ERK, p38, jun kinase and PKC-delta) and expression of stress proteins (focus: heat shock proteins; HSPs) important in the regulation of apoptosis; #3) NEP contributes to the regulation of these selected kinases and HSPs and promotes hypoxia-induced apoptosis of PA SMC by both peptidase-dependent and peptidase-independent mechanisms. Integrated experiments will be performed in wild type, NEP null and targeted NEP overexpressor mice, lung tissue and isolated PA SMC. Lentiviral constructs for wild type and mutant forms of NEP will be used to dissect peptidase-dependent vs novel peptidase-independent effects and provide new information on mechanisms that regulate susceptibility of PA SMC to apoptosis. These studies will also draw on unique mouse models of hypoxia-induced pulmonary vascular remodeling to increase our understanding of mechanisms that control susceptibility to chronic hypoxic PHTN and could identify new therapeutic targets to limit or reverse this important clinical problem. [unreadable] [unreadable]